Metabolic Engineering

Lignocellulosic ethanol has been considered as an alternative transportation fuel. Utilization of hemicellulosic fraction in lignocelluloses is crucial in economical production of lignocellulosic ethanol. However, this fraction has not efficiently been utilized by traditional yeast Saccharomyces cerevisiae. Genetically modified S. cerevisiae, which can utilize xylose, has several limitations including low ethanol yield, redox imbalance, and undesired metabolite formation similar to native xylose utilizing yeasts. Besides, xylose uptake is a major issue, where sugar transport system plays an important role. These genetically modified and wild-type yeast strains have further been engineered for improved xylose uptake. Various techniques have been employed to facilitate the xylose transportation in these strains. The present review is focused on the sugar transport machineries, mechanisms of xylose transport, limitations and how to deal with xylose transport for xylose assimilation in ...

Fatty acid metabolism is an attractive route to produce liquid transportation fuels and commodity oleochemicals from renewable feedstocks. Recently, genes and enzymes, which comprise metabolic pathways for producing fatty acid-derived compounds (e.g. esters, alkanes, olefins, ketones, alcohols, polyesters) have been elucidated and used in engineered microbial hosts. The resulting strains often generate products at low percentages of maximum theoretical yields, leaving significant room for metabolic engineering. Economically viable processes will require strains to approach theoretical yields, particularly for replacement of petroleum-derived fuels. This review will describe recent progress toward this goal, highlighting the scientific discoveries of each pathway, ongoing biochemical studies to understand each enzyme, and metabolic engineering strategies that are being used to improve strain performance.

A micropropagation system through leaf explant culture has been developed for Withania coagulans. Shoot bud proliferation occurred through both adventitious and de novo routes depending on the hormonal regime of the culture medium. Green compact nodular organogenic callus developed on Murashige and Skoog (MS) medium supplemented with 2.3 μM kinetin (Kn) and lower levels of 6–benzyladenine (BA) (13.3 μM) while multiple adventitious shoot bud differentiation occurred on medium fortified with 2.3 μM kinetin (Kn) and higher levels of BA (22.2 μM). Shoot buds were transferred to proliferation medium containing 2.2 μM BA, 2.3 μM Kn, and 3.9 μM phloroglucinol (PG) for further growth and development of shoot system. Elongated shoots were rooted using a two-step procedure involving pulse treatment of 7 days in a medium containing 71.6 μM choline chloride (CC) and 3.9 μM PG and then transferred to rooting medium containing ½ MS, 1.2 μM IBA, 3.6 μM PAA, and 14.3 μM CC for 3 weeks. Well-rooted plants were transferred to a greenhouse for hardening and further growth. Random amplification of polymorphic DNA (RAPD) showed monomorphic bands in all the plants thereby confirming clonality of the regenerants. Thin layer chromatography (TLC) showed the presence of withanolides in the regenerated plants. Quantification through reverse-phase HPLC revealed increased concentration of withanolides in the regenerated plants compared to the field-grown mother plant. Accumulation of withaferin A and withanolide A increased up to twofold and that of withanone up to tenfold. Direct regeneration via leaf explants will be useful for Agrobacterium-mediated genetic transformation, and will facilitate pathway manipulation using metabolic engineering for bioactive withanolides.

The Escherichia coli nar promoter is maximally induced under anaerobic conditions in the presence of nitrate ion or under anaerobic only conditions, depending on the genotype of the E. coli nar promoter. Previously, we found that the E. coli nar promoter has some desirable characteristics as an inducible promoter in the E. coli host strains. In this study, the E. coli nar promoter with lacZ gene at the downstream was cloned onto a broad-host-range Gram-negative vector, pBBR122. It was then induced in some other Gram-negative host strains, such as Agrobacterium, Pseudomonas, and Rhizobium, to determine whether the E. coli nar promoter could be used as an inducible promoter in these strains. From shake-flask experiments it was found that the wild-type E. coli nar promoter cloned onto pBBR122, pNW61, was suppressed under aerobic conditions in an Agrobacterium host strain, was partially induced under microaerobic only conditions, and was maximally induced under microaerobic conditions in the presence of nitrate ion. Whereas the mutant-type E. coli nar promoter cloned onto pBBR122, pNW618, was suppressed under aerobic conditions and was maximally induced under microaerobic conditions, regardless of the presence of nitrate ion. This kind of induction pattern observed for the E. coli nar promoters in the Agrobacterium host strain was similar to that observed for the E. coli nar promoters in the E. coli host strain. On the other hand, it was found that both of the E. coli nar promoters, pNW61 and pNW618, in a Pseudomonas host strain were partially induced under aerobic conditions and were maximally induced under microaerobic conditions, regardless of the presence of nitrate. Finally, it was found that both of the E. coli nar promoters in a Rhizobium host strain were minimally induced, regardless of the presence of oxygen or nitrate ion. Similar induction patterns for the three strains were also observed from fermentor experiments in which the dissolved oxygen (DO) level was tightly controlled. From an evolutionary point of view, the results from the three Gram-negative host strains indicate that the E. coli nar promoter system, including the promoter and regulatory proteins, was best conserved in the Agrobacterium host strain and the least conserved in the Rhizobium host strain. From an industrial point of view, the results indicate that the E. coli nar promoter system can be used as an oxygen-dependent inducible promoter in both Agrobacterium and Pseudomonas host strains.

The increased synthesis of building blocks of IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) through metabolic engineering is a way to enhance the production of carotenoids. Using E. coli as a host, IPP and DMAPP supply can be increased significantly through the introduction of foreign MVA (mevalonate) pathway into it. The MVA pathway is split into two parts with the top and bottom portions supplying mevalonate from acetyl-CoA, and IPP and DMAPP from mevalonate, respectively. The bottom portions of MVA pathway from Streptococcus pneumonia, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes and Saccharomyces cerevisiae were compared with exogenous mevalonate supplementation for ␤-carotene production in recombinant Escherichia coli harboring ␤-carotene synthesis genes. The E. coli harboring the bottom MVA pathway of S. pneumoniae produced the highest amount of ␤-carotene. The top portions of MVA pathway were also compared and the top MVA pathway of E. faecalis was found out to be the most efficient for mevalonate production in E. coli. The whole MVA pathway was constructed by combining the bottom and top portions of MVA pathway of S. pneumoniae and E. faecalis, respectively. The recombinant E. coli harboring the whole MVA pathway and ␤-carotene synthesis genes produced high amount of ␤-carotene even without exogenous mevalonate supplementation. When comparing various E. coli strains -MG1655, DH5␣, S17-1, XL1-Blue and BL21 -the DH5␣ was found to be the best ␤-carotene producer. Using glycerol as the carbon source for ␤-carotene production was found to be superior to glucose, galactose, xylose and maltose. The recombinant E. coli DH5␣ harboring the whole MVA pathway and ␤-carotene synthesis genes produced ␤-carotene of 465 mg/L at glycerol concentration of 2% (w/v).

Adaptive laboratory evolution is an important tool for the engineering of strains for industrially relevant phenotypes. Traditionally, adaptive laboratory evolution has been implemented to improve robustness of industrial strains under diverse operational conditions; however due to the required coupling between growth and survival, its application for increased production of secondary metabolites generally results in decreased production due to the metabolic burden imposed by, or toxicity of, the produced compound.

The industrial revolution, the first agricultural 'green revolution', and the development of antibiotics and therapeutic chemicals have brought significant and undeniable benefits to the human race. However, these advances demand high levels of energy, exploit natural resources and create large amounts of waste that creates an environmental burden for our planet. The pollution rate and character of many of the pollutants results in a rapid deterioration of the environment. Bioremediation functions to isolate and select microorganisms that operate under aerobic and anoxic conditions to remove these harmful pollutants. Current 'omics' technologies allow the exploitation of the catabolic potential of microbes without the need to cultivate them. Synthetic microbiology builds new catabolic pathways to remove recalcitrant pollutants from the environment.

Improving the quality and performance of soybean oil as biodiesel depends on the chemical composition of its fatty acids and requires an increase in monounsaturated acids and a reduction in polyunsaturated acids. Despite its current use as a source of biofuel, soybean oil contains an average of 25 % oleic acid and 13 % palmitic acid, which negatively impacts its oxidative stability and freezing point, causing a high rate of nitrogen oxide emission. Gas chromatography and ion mobility mass spectrometry were conducted on soybean fatty acids from metabolically engineered seed extracts to determine the nature of the structural oleic and palmitic acids. The soybean genes FAD2-1 and FatB were placed under the control of the 35SCaMV constitutive promoter, introduced to soybean embryonic axes by particle bombardment and downregulated using RNA interference technology. Results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 94.58 %) and a reduction in palmitic acid (to <3 %) in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and non-transgenic oil extracts.

Recent achievements have been made in the metabolic engineering of plant secondary metabolism. Various pathways have been altered using genes encoding biosynthetic enzymes or genes encoding regulatory proteins. In addition, antisense genes have been used to block competitive pathways, thereby increasing the flux towards the desired secondary metabolites.

Paclitaxel (trademark "Taxol") is a plant-derived isoprenoid natural product that exhibits potent anticancer activity. Taxol was originally isolated from the Pacific yew tree in 1967 and triggered an intense scientific and engineering venture to provide the compound reliably to cancer patients. The choices available for production include synthetic and biosynthetic routes (and combinations thereof). This chapter focuses on the currently utilized and emerging biosynthetic options for Taxol production. A particular emphasis is placed on the biosynthetic production hosts including macroscopic and unicellular plant species and more recent attempts to elucidate, transfer, and reconstitute the Taxol pathway within technically advanced microbial hosts. In so doing, we provide the reader with relevant background related to Taxol and more general information related to producing valuable, but structurally complex, natural products through biosynthetic strategies.

Hydrogen is an ideal, clean, and potentially sustainable energy carrier for the future due to its abundance and non-polluting nature. Numerous bacteria, cyanobacteria, and algae are capable of producing hydrogen from water, solar energy, and a variety of organic substrates. Improvement of these diverse biochemical pathways is needed in order to make biohydrogen competitive with current production methods. This review summarizes some of the main biological pathways that produce hydrogen and their limiting factors. It also describes how metabolic engineering strategies are being used to overcome these limitations, increase yields, and broaden substrate utilization.

Background: Vanillin is one of the most widely used flavouring agents, originally obtained from cured seed pods of the vanilla orchid Vanilla planifolia. Currently vanillin is mostly produced via chemical synthesis. A de novo synthetic pathway for heterologous vanillin production from glucose has recently been implemented in baker's yeast, Saccharamyces cerevisiae. In this study we aimed at engineering this vanillin cell factory towards improved productivity and thereby at developing an attractive alternative to chemical synthesis. Results: Expression of a glycosyltransferase from Arabidopsis thaliana in the vanillin producing S. cerevisiae strain served to decrease product toxicity. An in silico metabolic engineering strategy of this vanillin glucoside producing strain was designed using a set of stoichiometric modelling tools applied to the yeast genome-scale metabolic network. Two targets (PDC1 and GDH1) were selected for experimental verification resulting in four engineered strains. Three of the mutants showed up to 1.5 fold higher vanillin β-D-glucoside yield in batch mode, while continuous culture of the Δpdc1 mutant showed a 2-fold productivity improvement. This mutant presented a 5fold improvement in free vanillin production compared to the previous work on de novo vanillin biosynthesis in baker's yeast. Conclusion: Use of constraints corresponding to different physiological states was found to greatly influence the target predictions given minimization of metabolic adjustment (MOMA) as biological objective function. In vivo verification of the targets, selected based on their predicted metabolic adjustment, successfully led to overproducing strains. Overall, we propose and demonstrate a framework for in silico design and target selection for improving microbial cell factories.

Progress on plasmid-based (pDNA) vaccines requires simpler and efficient cultivation techniques for their production. A prevalent problem in the cultivation of Escherichia coli (the main host for pDNA vaccines production) is the aerobic production of acetate. In this work, a metabolically engineered Escherichia coli strain with strongly reduced acetate formation was tested for the production of a plasmid vaccine at

a b s t r a c t 2-methylpropan-1-ol (isobutanol) is a leading candidate biofuel for the replacement or supplementation of current fossil fuels. Recent work has demonstrated glucose to isobutanol conversion through a modified amino acid pathway in a recombinant organism. Although anaerobic conditions are required for an economically competitive process, only aerobic isobutanol production has been feasible due to an imbalance in cofactor utilization. Two of the pathway enzymes, ketol-acid reductoisomerase and alcohol dehydrogenase, require nicotinamide dinucleotide phosphate (NADPH); glycolysis, however, produces only nicotinamide dinucleotide (NADH). Here, we compare two solutions to this imbalance problem: (1) over-expression of pyridine nucleotide transhydrogenase PntAB and (2) construction of an NADH-dependent pathway, using engineered enzymes. We demonstrate that an NADH-dependent pathway enables anaerobic isobutanol production at 100% theoretical yield and at higher titer and productivity than both the NADPH-dependent pathway and transhydrogenase over-expressing strain. Our results show how engineering cofactor dependence can overcome a critical obstacle to nextgeneration biofuel commercialization.

Overexpression of genes in production pathways and permanent knockout of genes in competing pathways are often employed to improve production titer and yield in metabolic engineering. However, the deletion of a pathway responsible for growth and cell maintenance has not previously been employed, even if its competition with the production pathway is obvious. In order to optimize intracellular metabolism at each fermentation phase for bacterial growth and production, a methodology employing conditional knockout is required. We constructed a metabolic toggle switch in Escherichia coli as a novel conditional knockout approach and applied it to isopropanol production. The resulting redirection of excess carbon flux caused by interruption of the TCA cycle via switching gltA OFF improved isopropanol production titer and yield up to 3.7 and 3.1 times, respectively. This approach is a useful tool to redirect carbon flux responsible for bacterial growth and/or cell maintenance toward a synthetic production pathway.

Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However, recombinant expression in E. coli is not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic proteins and especially membrane proteins, linked to missing posttranslational modifications, proteolysis and aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration and development, but in some cases linked to complex culture media or processes. In this context, alternative bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have comparatively produced a "difficult-to-express" human protein, the lysosomal enzyme alpha-galactosidase A (hGLA) in E. coli and in the Electronic supplementary material The online version of this article (psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. haloplanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic instability and/or protein misfolding, the expression of hGLA gene in P. haloplanktis TAC125 allows obtaining active enzyme. These results are discussed in the context of emerging bacterial systems for protein production that represent appealing alternatives to the regular use of E. coli and also of more complex eukaryotic systems.

Ad oggi, grazie a numerosi studi, si conoscono molte proprietà relative agli esopolisaccaridi prodotti da microrganismi di varia natura. Grazie a tali proprietà, queste biomolecole trovano una grande applicazione in moltissimi settori industriali, tra cui quello alimentare. Gli importanti aspetti nutraceutici (come ad esempio effetti prebiotici, immunomodulatori, anti-tumorali, anti-infiammatori, anti-patogeni ecc..), la grande biosostenibilità ed ecocompatibilità, rendono gli esopolisaccaridi una risorsa preziosa sul cui studio e sulla cui produzione investire.

Plant metabolic engineering has the potential to provide for the needs of an expanding population. Environmentally benign biosyntheisis of novel materials and pharmaceutical proteins along with the opportunity to improve the productivity and nutritive value of crop plants has focused considerable effort towards the genetic manipulation of crop species. The most important output traits that could be conferred through biotechnology often require the coordinated expression of several foreign genes. Conservative estimates predict some 3000 proteins are posttranslationally imported into plant plastids. Among them are the enzymes of various metabolic pathways such as those involved in the biosynthesis of the tocopherols (vitamin E) and carotenoids (vitamin A), branched chain and aromatic amino acids, and fatty acids. The ability of the chloroplast to integrate and express foreign sequences as operons makes this site an attractive alternative for genetic manipulations. Multigene engineering, high levels of recombinant protein accumulation and the security of transgene containment due to maternal inheritance of plastid genomes in most crop species are some of the features that contribute to the potential of the chloroplast system. Here we offer an overview of the fundamental characteristics of plastid protein expression and consider some possible candidate genes for the improvement of crop species through metabolic engineering of pathways compartmentalized within plastids

Lactic acid bacteria (LAB) have a long tradition of use in the food industry, and the number and diversity of their applications has increased considerably over the years. Traditionally, process optimization for these applications involved both strain selection and trial and error. More recently, metabolic engineering has emerged as a discipline that focuses on the rational improvement of industrially useful strains. In the post-genomic era, metabolic engineering increasingly benefits from systems biology, an approach that combines mathematical modelling techniques with functional-genomics data to build models for biological interpretation and--ultimately--prediction. In this review, the industrial applications of LAB are mapped onto available global, genome-scale metabolic modelling techniques to evaluate the extent to which functional genomics and systems biology can live up to their industrial promise.

Progesteron adalah hormon pada tubuh, yang berfungsi  untuk mempertebal dinding rahim pada fasa luteal, dan menjaga keremajaan kulit. Kekurangan hormon progesteron dapat menyebabkan kemandulan,  kanker prostat, dan payudara.  Untuk mengatasi penyakit akibat kekurangan hormon progesteron,  dilakukan penelitian untuk  mencari bahan aktif yang memiliki struktur mirip dengan progesteron pada tumbuhan. Salah satunya ditemukan bahan aktif solasodin pada buah  Solanum khasianum. Senyawa ini diekstraksi dengan menggunakan pelarut etanol 70% dengan menggunakan bantuan sonikator yang divariasi intensitasnya 20%, 30%, 60% dan 100% selama 60 menit, kemudian dilanjutkan dengan hidrolisis menggunakan pelarut HCl 1N dalam metanol untuk isolasi senyawa solasodin. Setelah melalui proses isolasi, kristal hasil akhir di analisis dengan alat  fourier  transform infrared spectroscopy (FT-IR) dan metode titik leleh  untuk mengetahui kebenaran hasil ekstraksi berupa kristal solasodin. Dari 150 gram simplisia, didapat ekstrak solasodin terbanyak pada intensitas sonikasi 20% sebanyak 1,39 gram. Dengan intensitas optimal ini, dilakukan variasi waktu sonikasi selama 30 menit, 60 menit dan 120 menit. Hasil sonikasi selama 60 menit menghasilkan solasodin yang terbanyak yaitu 1,39 gram. Hasil karakterisasi FT-IR menunjukkan fingerprint struktur solasodin yang terdegradasi gugus C-OH menjadi C=O. Hasil pengujian titik leleh menunjukkan kristal meleleh pada suhu 202,61 0C.

Carotenoids are indispensable to plants and play a critical role in human nutrition and health. Significant progress has been made in our understanding of carotenoid metabolism in plants. The biosynthetic pathway has been extensively studied. Nearly all the genes encoding the biosynthetic enzymes have been isolated and characterized from various organisms. In recent years, there is an increasing body of work on the signaling pathways and plastid development, which might provide global control of carotenoid biosynthesis and accumulation. Herein, we will highlight recent progress on the biosynthesis, regulation, and metabolic engineering of carotenoids in plants, as well as the future research towards elucidating the regulatory mechanisms and metabolic network that control carotenoid metabolism.

Polyhydroxyalkanoates have gained major importance due to their structural diversity and close analogy to plastics. These are gaining more and more importance world over. Different sources (natural isolates, recombinant bacteria, plants) and other methods are being investigated to exert more control over the quality, quantity and economics of poly(3-hydroxybutyrate) (PHB) production. Their biodegradability makes them extremely desirable substitutes for synthetic plastics. The PHB biosynthetic genes phbA, phbB and phbC are clustered and organized in one phbCAB operon. The PHB pathway is highly divergent in the bacterial genera with regard to orientation and clustering of genes involved. Inspite of this the enzymes display a high degree of sequence conservation. But how similar are the mechanisms of regulation of these divergent operons is as yet unknown. Structural studies will further improve our understanding of the mechanism of action of these enzymes and aid us in improving and selecting better candidates for increased production. Metabolic engineering thereafter promises to bring a feasible solution for the production of ''green plastic''.

Here, we report the engineering of the industrially relevant Corynebacterium glutamicum for putrescine production. C. glutamicum grew well in the presence of up to 500 mM of putrescine. A reduction of the growth rate by 34% and of biomass formation by 39% was observed at 750 mM of putrescine. C. glutamicum was enabled to produce putrescine by heterologous expression of genes encoding enzymes of the arginine-and ornithine decarboxylase pathways from Escherichia coli. The results showed that the putrescine yield by recombinant C. glutamicum strains provided with the arginine-decarboxylase pathway was 40 times lower than the yield by strains provided with the ornithine decarboxylase pathway. The highest production efficiency was reached by overexpression of speC, encoding the ornithine decarboxylase from E. coli, in combination with chromosomal deletion of genes encoding the arginine repressor ArgR and the ornithine carbamoyltransferase ArgF. In shake-flask batch cultures this strain produced putrescine up to 6 g/L with a space time yield of 0.1 g/L/h. The overall product yield was about 24 mol% (0.12 g/g of glucose).

Shilajit moomiyo is an adaptogenic herb which can be used to enhance physical and mental performance. Shilajit is believed to amplify the benefits of other herbs by enhancing their bio-availability. Shilajit is also believed to increase protein synthesis, growth rate and/or energy intake. Suitably, the nutritional composition can include a Shilajit material or extract which has been standardized to include or contain 10% humic acid and/or 10% fulvic acid. In accordance with certain other embodiments, the nutritional composition can alternatively and/or additionally contain fulvic acid, humic acid, and/or another botanical, natural, or herbal source of fulvic acid and/or humic acid. Shilajit plays an important part in boosting the energy metabolism process in the body.

Camptothecin (CPT) is a modified monoterpene indole alkaloid produced by Camptotheca acuminata (Nyssaceae), Nothapodytes foetida, Pyrenacantha klaineana, Merrilliodendron megacarpum (Icacinaceae), Ophiorrhiza pumila (Rubiaceae), Ervatamia heyneana (Apocynaceae) and Mostuea brunonis (Gelsemiaceae), species belonging to unrelated orders of angiosperms. From the distribution of CPT and other secondary metabolites, it has been postulated that the genes encoding enzymes involved in their biosynthesis evolved early during evolution. These genes were presumably not lost during evolution but might have been ''switched off'' during a certain period of time and ''switched on'' again at some later point. The CPT derivatives, irinotecan and topotecan, are used throughout the world for the treatment of various cancers, and over a dozen more CPT analogues are currently at various stages of clinical development. The worldwide market size of irinotecan/topotecan in 2002 was estimated at about $750 million and at $1 billion by 2003. In spite of the rapid growth of the market, CPT is still harvested by extraction from bark and seeds of C. acuminata and N. foetida. All parts of C. acuminata contain some CPT, although the highest level is found in young leaves ($4-5 mg g À1 dry weight), approximately 50% higher than in seeds and 250% higher than in bark. The development of hairy root cultures of O. pumila and C. acuminata, and the cloning and characterization of genes encoding key enzymes of the pathway leading to CPT formation in plants has opened new possibilities to propose alternative and more sustainable production systems for this important alkaloid.

Significant advances in plant cell, tissue and organ culture (PCTOC) have been made in the last five decades. PCTOC is now thought to be the underlying technique for understanding general or specific biological functions of the plant kingdom, and it is one of the most flexible foundations for morphological, physiological and molecular biological applications of plants. Furthermore, the recent advances in the field of information technology (IT) have enabled access to a large amount of information regarding all aspects of plant biology. For example, sequencing information is stored in mega repositories such as the National Center for Biotechnology Information (NCBI), which can be easily accessed by researchers worldwide. To date, the PCTOC and IT combination strategy for regulation of target plant metabolism and the utilization of bioactive plant metabolites for commercial purposes is essential. In this review, the advantages and the limitations of these methodologies, especially reg...

Acrylic acid might become an important target for fermentative production from sugars on bulk industrial scale, as an alternative to the current petrochemical production. Metabolic engineering approaches will be required to develop a host microorganism that may enable such a fermentation process. Hypothetical metabolic pathways for insertion into a host organism are discussed. The pathway should have plausible mass and redox balances, plausible biochemistry and plausible energetics, while giving the theoretically maximum yield of acrylate on glucose without using aeration or adding electron acceptors. Candidate metabolic pathways that might lead to the theoretically maximum yield run via -alanine, methylcitrate or methylmalonate-CoA. The energetics and enzymology of these pathways, including product excretion, should be studied in more detail to confirm this. The expression of a selected pathway in a host organism will require extensive genetic engineering.

The critical stages during the growth of crops are the uniform seed germination, early seedling growth, and uniform plant stand. Low crop yield is attributed to uneven seed germination and seedling growth. Therefore, the quality of seed can be improved through priming in addition to the field management techniques for better seed germination. Priming is a physiological technique of seed hydration
and drying to enhance the pregerminative metabolic process for rapid germination, seedling growth, and final yield under normal as well as stressed conditions. The primed seeds show faster and uniform seed germination due to different enzyme activation, metabolic activities, biochemical process of cell repair, protein synthesis, and improvement of the antioxidant defense system as compared to unprimed
seeds. There are many techniques of seed priming which are broadly divided into conventional methods (hydro-priming, osmo-priming, nutrient priming, chemical priming, bio-priming, and priming with plant growth regulators) and advanced methods (nano-priming and priming with physical agents). However, priming is strongly affected by various factors such as temperature, aeration, light, priming duration, and seed characteristics. This chapter highlights the priming mechanism
and the available technologies as a tool for superficial seed germination and crop stand. An experiment with reference to the importance of priming toward vigor seed germination and seedling growth was conducted, and its results have been added in this chapter.

El ácido succínico producido por diversos microorganismos se puede utilizar como un precursor de muchos productos químicos industrialmente importantes en las industrias alimentarias, químicas y farmacéuticas. La evaluación de costo de materia prima y la estimación del tamaño potencial de mercado indican claramente que el proceso de ácido succínico a base de petróleo actual se sustituye por el sistema de producción de ácido succínico fermentativo en el futuro previsible. Este artículo revisa los procesos para la producción de ácido succínico fermentativo, con especial énfasis en el uso de varios productores de ácido succínico prometedoras incluyendo succinogenes actinomycetemcomitans, succiniciproducens Anaerobiospirillum, succiniciproducens Mannheimia y Escherichia coli recombinante. Los procesos para la recuperación de ácido succínico de caldo de fermentación también se revisan brevemente. Por último, le sugerimos otras obras necesarias para mejorar el rendimiento cepa adecuada para la adecuada comercialización de la producción de ácido succínico fermentativo.

Approximately 80% of the world inhabitants depend on the medicinal plants in the form of traditional formulations for their primary health care system well as in the treatment of a number of diseases since the ancient time. Many commercially used drugs have come from the information of indigenous knowledge of plants and their folk uses. Linking of the indigenous knowledge of medicinal plants to modern research activities provides a new reliable approach, for the discovery of novel drugs much more effectively than with random collection. Increase in population and increasing demand of plant products along with illegal trade are causing depletion of medicinal plants and many are threatened in natural habitat. Plant tissue culture technique has proved potential alternative for the production of desirable bioactive components from plants, to produce the enough amounts of plant material that is needed and for the conservation of threatened species. Different plant tissue culture systems ...

Nitrogenase a b s t r a c t Biological hydrogen production processes offer a technique through which renewable energy sources like biomass can be utilized for the generation of the cleanest energy carrier for the use of mankind. Hydrogen intensive research work has already been carried out on the advancement of these processes, such as the development of genetically modified microorganism, metabolic engineering, improvement of the reactor designs, use of different solid matrices for the immobilization of whole cells, biochemical assisted bioreactor, development of two-stage processes, etc. for higher H 2 -production rates. Maximum H 2 yield is found to be 7.1 mol H 2 /mol glucose. However, major bottlenecks for the commercialization of these processes are lower H 2 yield and rate of H 2 production. Suitable microbial cultures are required to handle waste materials efficiently, which are usually complex in nature. This will serve dual purposes: clean energy generation and bioremediation. Scale-up studies on fermentative H 2 -production processes have been done successfully. Pilot plant trials of the photo-fermentation processes require more attention.

Astaxanthin is a red xanthophyll (oxygenated carotenoid) with large importance in the aquaculture, pharmaceutical, and food industries. The green alga Haematococcus pluvialis and the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous are currently known as the main microorganisms useful for astaxanthin production at the industrial scale. The improvement of astaxanthin titer by microbial fermentation is a requirement to be competitive with the synthetic manufacture by chemical procedures, which at present is the major source in the market. In this review, we show how the isolation of new strains of X. dendrorhous from the environment, the selection of mutants by the classical methods of random mutation and screening, and the rational metabolic engineering, have provided improved strains with higher astaxanthin productivity. To reduce production costs and enhance competitiveness from an industrial point of view, low-cost raw materials from industrial and agricultural origin have been adopted to get the maximal astaxanthin productivity. Finally, fermentation parameters have been studied in depth, both at flask and fermenter scales, to get maximal astaxanthin titers of 4.7 mg/g dry cell matter (420 mg/l) when X. dendrorhous was fermented under continuous white light. The industrial scale-up of this biotechnological process will provide a cost-effective method, alternative to synthetic astaxanthin, for the commercial exploitation of the expensive astaxanthin (about $2,500 per kilogram of pure astaxanthin).

Recombinant DNA (rDNA) technologies (genetic, protein, and metabolic engineering) allow the production of a wide range of peptides, proteins, and biochemicals from naturally nonproducing cells. These technologies, now approx 25 yr old, have become one of the most important technologies developed in the twentieth century. Pharmaceutical products and industrial enzymes were the first biotech products on the world market made by means of rDNA. Despite important advances in rDNA applications in mammalian cells, yeasts still represent attractive hosts for the production of heterologous proteins. In this review we summarize advantages and limitations of the main and most promising yeast hosts.

Agricultural biotechnology offers a robust series of tools by which to address global concerns such as food security, crop protection, and fuel/energy requirements. A number of advances made recently in plant molecular biology also have resulted in applications which largely focus on improving global human health. This review describes some of the recent innovations in plant biotechnology that have come to the forefront over the past year. Included are novel techniques by which plants can be improved as platforms for biopharmaceutical protein production, a growing field also referred to as 'molecular pharming'. The metabolic engineering of plants to produce compounds which have additional nutritional benefits is also outlined. The review concludes with a discussion of the future impact that these innovations may have both on global health and on the development of our future intellectual property landscape.

Microalgae are autotrophic microorganisms having extremely high photosynthetic efficiency and are valued as rich source of lipids, hydrocarbons, and other complex oils for biodiesel besides being an invaluable source of bioethanol, biomethane, and biohydrogen. Biodiesel produced from oilseed crops such as jatropha and soy have lower yields per unit land area and threaten food security. Indeed, microalgae have higher oil yields amounting to about 40 times more oil per unit area of land in comparison to terrestrial oilseed crops such as soy and canola. Further, microalgae production does not require arable land for cultivation. Biofuel is regarded as a proven clean energy source and several entrepreneurs are attempting to commercialize this renewable source. Technology for producing and using biofuel has been known for several years and is frequently modified and upgraded. In view of this, a review is presented on microalgae as second generation biofuel. Microalgal farming for biomass production is the biggest challenge and opportunity for the biofuel industry. These are considered to be more efficient in converting solar energy into chemical energy and are amongst the most efficient photosynthetic plants on earth. Microalgae have simple cellular structure, a lipid-rich composition, and a rapid rate of reproduction. Many microalgal strains can be grown in saltwater and other harsh conditions. Some autotrophic microalgae can also be converted to heterotrophic ones to accumulate high quality oils using organic carbon. However, there are several technical challenges that need to be addressed to make microalgal biofuel profitable. The efficiency of microalgal biomass production is highly influenced by environmental conditions, e.g., light of proper intensity and wavelength, temperature, CO2 concentration, nutrient composition, salinities and mixing conditions, and by the choice of cultivation systems: open versus closed pond systems, photobioreactors. Currently, microalgae for commercial purpose are grown mostly in open circular/elongated “raceway” ponds which generally have low yields and high production costs. However, a hybrid system combining closed photobioreactor and open pond is a better option. The biggest hurdle in commercialization of microalgal biofuel is the high cost and energy requirement for the microalgal biomass production, particularly agitation, harvesting, and drying of biomass. In order to conserve energy and reduce costs, algae are often harvested in a two-step process involving flocculation followed by centrifugation, filtration, or micro-straining to get a solid concentration. However, the major bottlenecks in algal biodiesel production within the cell can be identified and handled by adopting a system approach involving transcriptomics, proteomics, and metabolomics. Research and developments in the field of new materials and advanced designs for cultivation in closed bioreactors, use of waste water for biomass production, screening of efficient strains, high-value coproduct strategy, and cutting-edge metabolic engineering are thought to provide the biggest opportunities to substantially improve the cost effectiveness of such production systems.